Synergistic inhibition of human α-1,3-fucosyltransferase V

Article abstract of DOI:10.1021/ja960274f

Human α-1,3-fucosyltransferase V (FucT V), which catalyzes the transfer of L-fucose moiety from guanosine diphosphate β-L-fucose (GDP-Fuc) to an acceptor sugar to form sialyl Lewis x (sLe(x)), was shown to proceed through an ordered, sequential mechanism

Full text of DOI:10.1021/ja960274f

J. Am. Chem. Soc. 1996, 118, 7653-7662
7653
Synergistic Inhibition of Human R-1,3-Fucosyltransferase V
Lei Qiao,† Brion W. Murray,† Makoto Shimazaki,† Jody Schultz,‡ and
Chi-Huey Wong*,†
Contribution from the Department of Chemistry, The Scripps Research Institute,
10666 North Torrey Pines Road, La Jolla, California, 92037, and Cytel Corporation,
3525 John Hopkins Court, San Diego, California, 92121
ReceiVed January 25, 1996X
Abstract: Human R-1,3-fucosyltransferase V (FucT V), which catalyzes the transfer of L-fucose moiety from guanosine
diphosphate â-L-fucose (GDP-Fuc) to an acceptor sugar to form sialyl Lewis x (sLex), was shown to proceed through
an ordered, sequential mechanism by product inhibition studies. The designed azatrisaccharide propyl 2-acetamido-
2-deoxy-4-O-(â-D-galactopyranosyl)-3-O-(2-(N-(â-L-homofuconojirimycinyl))ethyl)-R-D-glucopyranoside (2), prepared
by covalently linking the N-group of â-L-homofuconojirimycin (1) to the 3-OH of LacNAc through an ethylene
unit, in the presence of GDP was found to be an effective inhibitor of FucT V. In the presence of 30 µM GDP, the
concentration of 2 necessary to cause 50% inhibition was reduced 77-fold to 31 µM. Presumably, the azatrisaccharide
and GDP form a complex which mimics the transition state of the enzymatic reaction. Given the low affinity of
FucT V for its substrate LacNAc (Km ) 35 mM), the designed azatrisaccharide in the presence of GDP represents
the most potent synergistic inhibitor complex reported so far.
Introduction
(sLex, Figure 1), a ligand for E-selectin involved in inflammatory
process and tumor development.7 This enzyme has been studied
for acceptor specificity and used in the chemoenzymatic
synthesis of sLex.8,9 This enzyme accepts both N-acetyllac-
tosamine (LacNAc) and sialyl LacNAc as the substrates with
Km values of 35 and 100 mM, respectively. The related enzyme
R-1,2-fucosyltransferase has been hypothesized to proceed
through a simple ion-pair transition state which results in the
inversion of anomeric configuration,10 R-1,3-fucosyltransferases
may operate Via a similar mechanism involving a displacement
of GDP by the acceptor hydroxyl group assisted by a base on
the enzyme (Figure 2). To date there is no X-ray crystal
structure of any glycosyltransferase reported and none of the
glycosyltransferases have been studied in detail with respect to
their mechanism, though the kinetic mechanisms of a limited
Many antigenic oligosaccharides on the cell surface are
fucosylated. These fucose-containing structures are regarded
as oncodevelopmental antigens since they accumulate in a large
variety of human cancers.1 The biosynthesis of these structures
requires the action of several glycosyltransferases, of which
fucosylation by a class of fucosyltransferases (FucT) is the last
and critical step.2 R-L-Fucosidase3 is a degradative enzyme
which is involved in the hydrolytic removal of fucose residue
from these glycoconjugates. Studies have indicated that in-
creased activities of fucosyltransferase and R-L-fucosidase are
responsible for the abnormal expression of these fucose-
containing antigens in endometrial carcinoma.4 Therefore,
inhibitors of Fuc-T and R-fucosidase are potentially useful as
anti-inflammatory and anti-tumor agents.
Fucosyltransferases catalyze the transfer of the L-fucose
moiety from guanosine diphosphate â-L-fucose (GDP-Fuc) to
the corresponding glycoconjugate acceptors to form an R-1,2,
R-1,3/4, R-1,3 or R-1,6 linkage.5 Five human R-1,3-fucosyl-
transferases have been cloned and mapped on chromosomes;6
among them, R-1,3-fucosyltransferase V (FucT V) has been
shown to be responsible for the production of sialyl Lewis x
(6) (a) de Vries, T.; Srnka, C. A.; Palcic, M. M.; Swiedler, S. J.; van
den Eijnden, D. H.; Macher, B. A. J. Biol. Chem. 1995, 270, 8712. (b)
McCurley, R. S.; Recinos III, A.; Olsen, A. S.; Gingrich, J. C.; Szczepaniak,
D.; Cameron, H. S.; Krauss, R.; Weston, B. W. Genomics 1995, 26, 142.
(c) Kukowska-Latallo, J. F.; Larsen, R. D.; Nair, R. P.; Lowe, J. B. Genes
DeV. 1990, 4, 1288. (d) Natsuka, S.; Gersten, K. M.; Zenita, K.; Kannagi,
R.; Lowe, J. B. J. Biol. Chem. 1994, 269, 16789. (e) Sasaki, K. Kurata, K.;
Funayama, K.; Nagata, M.; Watanabe, E.; Ohta, S.; Hanai, N.; Nishi, T. J.
Biol. Chem. 269, 14730. (f) Lowe, J. B.; Kukowska-Latallo, J. F.; Nair, R.
P.; Larsen, R. D.; Marks, R. M.; Macher, B. A.; Kelly, R. J.; Ernst, L. K.
J. Biol. Chem. 1991, 266, 17467. (g) Goelz, S. E.; Hession, C.; Goff, D.;
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1990, 63, 1349. (h) Kumar, R.; Potvin, B.; Muller, W. A.; Stanley,
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R. D.; Lowe, J. B. J. Biol. Chem. 1992, 267, 4152. (j) Weston, B. W.;
Smith, P. L.; Kelly, R. J.; Lowe, J. B. J. Biol. Chem. 1992, 267, 24575. (k)
Koszdin, K. L.; Bowen, B. R. Biochem. Biophys. Res. Commun. 1992, 187,
152.
† The Scripps Research Institute.
‡ Cytel Corp.
X Abstract published in AdVance ACS Abstracts, August 1, 1996.
(1) (a) Hakomori, S. AdV. Cancer Res. 1989, 52, 257. (b) Hakomori, S.;
Nudelman, E.; Levery, S. B.; Kannagi, R. J. Biol. Chem. 1984, 259, 4672.
(c) Feizi, T. Nature 1985, 314, 53.
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(b) Holme, E. H.; Ostrander, G. K.; Hakomori, S. J. Biol. Chem. 1986,
261, 3737. (c) Hanisch, F. G.; Mitsakos, A.; Schroten, H.; Uhlenbruck, G.
Carbohydr. Res. 1988, 178, 23. (d) Kornfeld, R.; Kornfeld, S. Annu. ReV.
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S0002-7863(96)00274-0 CCC: $12.00 © 1996 American Chemical Society

7654 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Qiao et al.
that these aza sugars synergistically inhibit fucosyltransferase
in the presence of GDP.8,9 Since both glycosidation and
glycosytransfer reactions are believed to involve transition states
with substantial oxocarbenium ion character, it has been
proposed that the protonated aza sugars may mimic the charge
distribution of the glycosyl cation developed in the transition
state.17 The synergistic inhibition, on the other hand, indicates
a possible interaction of aza sugars, GDP, and the acceptor sugar
in the active site of the enzyme to mimic the transition state of
the fucosyl transfer reaction (Figure 3). One advantage of using
this type of synergistic inhibition is that a relatively high
concentration of GDP is present in cells. The cellular concen-
tration of GTP has been reported to be 0.3-0.5 mM in human
cell lines18 and that of GDP is 5-10% of the GTP concentra-
tion.19 This translates into a 0.015-0.050 mM cellular con-
centration of GDP in these human cell lines, which argues for
the in ViVo relevance of the synergism between GDP and aza
sugars in the inhibition of FucT V. A similar strategy has been
used in the inhibition of UDP-GlcNAc enolpyruvoyl transferase
of the antibiotic fosfomycin,20 which requires the presence of
UDP-GlcNAc in ViVo for synergistic inhibition. Monosaccha-
ride aza sugar inhibitors are, however, not specific because they
only interfere with the binding of sugar nucleotide to the
enzyme, not with the acceptor substrate which determines the
specificity of the enzyme.
Figure 1. Structure of sialyl Lewis x. R-1,3-Fucosyltransferase V
catalyzes the addition of fucose to LacNAc or sialyl LacNAc.
number of enzymes have been studied.11-14 For example, â-1,4-
galactosyltransferase,12 â-D-xylosyltransferase,13 and â-1,2-N-
acetylglucosaminyl transferase II14 have been reported to
catalyze by an ordered, sequential manner while R-1,2-fuco-
syltransferase and UDP-glucuronosyltransferase are proposed
to have a random binding mechanism.10,11c The kinetic mech-
anism of FucT V has not been reported to date.
The catalytic residues responsible for abstraction of the proton
from the acceptor OH in most glycosyltransfer reactions re-
main elusive. Oligosaccharyl transferase has been shown to
have a catalytic base with a pKa ) 6.0 which was suggested to
be a histidine residue.11c The enzyme was found to be un-
stable at pH values greater than its optimal pH, 7.0. Most
galactosyl transferases are also active in the pH range from 6
to 8.11e Glycosyltransferases require metal cofactors, typically
manganese.11a,c,d,13,14 The divalent manganese cofactor of â-1,4-
galactosyltransferase has been shown to bind to the enzyme
before the donor nucleotide sugar, UDP-galactose, and release
from the galactosyltransferase in the form of a Mn2+‚UDP
complex.11a
To date only limited success has been achieved in the
development of inhibitors of these enzymes. Most efforts have
focused on the production of unreactive structural analogs of
GDP-Fuc.15 A bisubstrate inhibitor of R-1,2-fucosyltransferase
containing the acceptor structure and GDP portion of the donor
substrate has also been reported.10 However, these approaches
only produced inhibitors with either comparable or slightly
increased activity compared to that of the product inhibitor GDP.
Very recently, trisubstrate analogs of R-1,3-fucosyltransferase
containing D-glucose or N-acetyl-D-glucosamine, L-fucose ana-
logs, and GDP have been synthesized as potential inhibitors,
but no inhibition analysis was reported.16
Our approach to the construction of fucosyltransferase-
specific inhibitors is based on mimicking the proposed transition
state of the fucosyl transfer reaction by covalently linking an
aza sugar to the acceptor substrate. A designed inhibitor is
shown in Figure 4 in which â-L-homofuconojirimycin (1),21
a
potent inhibitor of R-fucosidase (Ki ) 5.6 nM), is linked to the
3-position of the acceptor substrate LacNAc Via an ethylene
spacer to form the azatrisaccharide 2. This azatrisaccharide
could then form a complex with GDP to mimic the transition
state of the enzymatic reaction and thereby inhibit the enzyme
in a sequence-specific, synergistic manner. The ethylene spacer
is used to mimic the partially-forming glycosidic linkage; the
flexibility of the linker may allow the aza analog of L-fucose
and LacNAc residues to adopt the correct relative orientation
for maximal enzymatic recognition. In this paper, we reported
our kinetic investigation studies of FucT V, chemoenzymatic
synthesis of inhibitors 1 and 2 for the inhibition studies of FucT
V, and elucidation of the enzyme mechanism.
Results and Discussion
Kinetic Mechanism of FucT V. The product inhibition
studies were conducted with purified FucT V. The inhibition
mode was determined by inspection of the inhibition pattern of
the double reciprocal analysis and statistical analysis of the
nonlinear, least squares fit of the data to the equations for
competitive, noncompetitive, and uncompetitive inhibition (see
the Experimental Section). Double reciprocal analysis of FucT
V as a function of GDP and GDP-Fuc exhibited a competitive
pattern. Analysis of the data with the Compo FORTRAN
A number of aza sugars have been found to be inhibitors of
glycosidases and glycosyltransferases. Certain aza sugars are
not only inhibitors of R-fucosidase but also moderate inhibitors
of fucosyltransferases; in addition, preliminary studies indicate
(11) (a) Tsopanakis, A. D.; Herries, D. G. Eur. J. Biochem. 1978, 83,
179-188. (b) Nakazawa, K.; Furukawa, K.; Narimatsu, K.; Kobata, A. J.
Biochem. 1993, 113, 747-753. (c) Yin, H.; Bennett, G.; Jones, J. P. Chem.-
Biol. Interactions 1994, 90, 47-58. (d) Breuer, W.; Bause, E. Eur. J.
Biochem. 1995, 228, 689-696. (e) Ram, B. P.; Munjal, D. D. CRC Crit.
ReV. Biochem. 1987, 17, 257-311.
(12) (a) Khatra, B. S.; Herries, D. G.; Brew, K. Eur. J. Biochem. 1974,
44, 537. (b) Kim, C. K.; Singh, A. N.; Raushel, F. M. Arch. Biochem.
Biophys. 1988, 267, 54-59. (c) Ats, S.-C; Lehmann, J.; Petry, S. Carbohydr.
Res. 1992, 233, 125-139.
(13) Kearns, A. E.; Campbell, S. C.; Westley, J.; Schwartz, N. B.
Biochemistry 1991, 30, 7477.
(17) (a) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y; Kajimoto, T. Angew.
Chem. Int. Ed. Engl. 1995, 34, 521. (b) Jeong, J.-H.; Murray, B. W.;
Takayama, S.; Wong, C.-H. J. Am. Chem. Soc., in press. (c) Ganem, B.
Acc. Chem. Res., in press. (d) Look, G. C.; Fotsch, C. H.; Wong, C.-H.
Acc. Chem. Res. 1993, 26, 182. (e) Winchester, B.; Fleet, G. W. J.
Glycobiology 1992, 2, 199.
(14) Bendiak, B.; Schachter, H. J. Biol. Chem. 1987, 262, 5784.
(15) (a) Cai, S.; Stroud, M. R.; Hakomori, S.; Toyokuni, T. J. Org. Chem.
1992, 57, 6693. (b) Luengo, J. T.; Gleason, J. G. Tetrahedron Lett. 1992,
33, 6911.
(16) (a) Heskamp, B. M.; Veeneman, G. H.; van der Marel, G. A.; van
Boeckel, C. A. A.; van Boom, J. H. Tetrahedron 1995, 51, 8397. (b)
Heskamp, B. M.; van der Marel, G. A.; van Boom, J. H. J. Carbohydr.
Chem. 1995, 14, 1265.
(18) Yabaluri, N.; Medaihradsky, F. Mol. Pharmacol. 1995, 48, 690.
(19) Gamberucci, A.; Innocenti, B.; Fulceri, R.; Banhegyi, G.; Giunti,
R.; Poxxan, T.; Benedetii, A. J. Biol. Chem. 1994, 269, 23597.
(20) Marquardt, J. L.; Brown, E. D.; Lane, W. S.; Haley, T. M.; Ichikawa,
Y.; Wong, C.-H.; Walsh, C. T. Biochemistry 1994, 33, 10646-10651.
(21) For chemical synthesis, see: Fleet, G. W. J.; Namgoong, S. K.;
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30, 4439.

Inhibition of R-1,3-Fucosyltransferase V
J. Am. Chem. Soc., Vol. 118, No. 33, 1996 7655
Figure 2. Proposed mechanisms of hydrolysis by fucosidase and fucosylation by R-1,3-fucosyltransferase V.
program22 yielded a Ki of 29 µM for GDP. The fucosylation
product Lewis x (Lex) was found to be a moderate inhibitor
with a Ki ) 52.3 mM as determined by Dixon analysis. Lex-
â-O-(CH2)5CO2CH323 was found to be a noncompetitive inhibi-
tor with respect to LacNAc and a noncompetitive inhibitor with
respect to GDP-Fuc. FucT V was reported to be subject to
potent substrate inhibition by GDP-fucose at concentrations
greater than 0.2 mM in the presence of 10 mM LacNAc.9 These
results, coupled with the reported noncompetitive inhibition
mode of GDP9 with respect to LacNAc (Kii ) 0.12 mM, Kis )
0.16 mM), are consistent with an ordered, sequential mecha-
nism24 (Figure 5). The general rate equations for an ordered,
Figure 3. Proposed model for the synergistic inhibition of R-1,3-
fucosyltransferase V by the combination of an aza sugar and GDP.
sequential, BiBi mechanism including product inhibition terms
have been described (eqs 1 and 2)25 Equation 1 describes the
acetal (3). Hydrogenation of 3 catalyzed by nickel (P2-Ni)27
generated in situ gave the cis-olefin 4 (81%), which was then
oxidized with m-CPBA to give epoxide 5 (71%). Stereoselec-
tive azide opening28 of the epoxide followed by acidic hydrolysis
provided the (()-threo-azidoaldehyde 6. FDP aldolase-
catalyzed aldol condensation of 6 with dihydroxyacetone
phosphate29 (DHAP) followed by dephosphorylation with acid
phosphatase afforded the desired enantiomerically pure azi-
doketose 7 (66%). Hydrogenation of 7 in the presence of Pd/C
produced â-L-homofuconojirimycin (1) as the only product
(100%) as shown by NMR spectroscopy. The high stereose-
lectivity of the hydrogenation is consistent with the early
observations that hydrogen is delivered from the less hindered
side of the possible imine intermediate during the reductive
amination process.17d
The convergent strategy for the synthesis of compound 2
involved a coupling of protected 1 and a LacNAc derivative.
Benzylation of 1 with benzyl bromide and sodium hydride
generated the fully benzylated compound 8 (Scheme 2). The
well-resolved spectrum of 8 combined with proton decoupling
experiments allowed the unequivocal determination of the aza
sugar configuration. The pertinent coupling constants, J2,3 (8.0
Hz), J3,4 (8.0 Hz), and J4,5 (2.9 Hz), clearly indicate the trans-
diaxial orientation between H 2-3, and H 3-4, as well as the
V ) VmaxA/{Km,A(1 + Q/Ki,Q)(1 + Ki,AKm,B/Km,AB) +
A(1 + Km,B/B)} (1a)
V ) VmaxB/{Km,B[1 + (Ki,A/A)(1 + Q/Ki,Q)] +
B[1 + (Km,A/A)(1 + Q/Ki,Q)]} (1b)
velocity (V) in the presence of only GDP, eq 1a as a function
of GDP-fucose concentration, and eq 1b as a function of Lac-
NAc concentration. Vmax is the maximal initial velocity, Ki,A
is an inhibition constant, and Km,A and Km,B are Michaelis con-
stants. Equation 2 is the rate equation for the velocity in the
presence of only Lewis x, eq 2a as a function of GDP-fucose
concentration, and eq 2b as a function of LacNAc concentration.
V ) VmaxA/{Km,A[1 + (Ki,AKm,B/Km,AB)(1 + P/Ki,Q)] +
A[1 + (Km,B/B)(1 + P/Ki,Q) + P/Ki,P]} (2a)
V ) VmaxB/{Km,B[(1 + Ki,A/A)(1 + P/Ki,Q)] +
B[1 + Km,B/A + P/Ki,P]} (2b)
A ) [GDP-fucose]
P ) [Lewis x]
B ) [LacNAc]
Q ) [GDP]
Synthesis of 1 and 2. â-L-Homofuconojirimycin (1) was
prepared conveniently by an aldolase-based strategy26 as
shown in Scheme 1. The acceptor substrate for aldolase was
synthesized from commercially available 2-butyn-1-al diethyl
(25) Segal, I. H. In Enzyme Kinetics, BehaVior and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems; John Wiley and Sons, Inc.:
New York, 1975; p 560-590.
(26) For a recent review for aldolase-catalyzed reactions see: Gijsen,
H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. ReV. 1996, 96, 4442-
473.
(22) Cleland, W. W. Method Enzymol. 1979, 63, 103.
(23) Lin, C.-C.; Shimazaki, M.; Heck, M.-P.; Aoki, S.; Wang, R.; Kimura,
T.; Ritzen, H.; Takayama, S.; Wu, S.-H.; Weitz-Schmidt, G.; Wong, C.-H.
J. Am. Chem. Soc., submitted.
(24) (a) Cleland, W. W. The Enzymes, 3rd ed.; Academic Press: New
York, 1970; Vol. 2, pp 1-65. (b) Goldsmith, J. O.; Kuo, K. C. J. Biol.
Chem. 1993, 268, 18485.
(27) Brown, C. A.; Ahuja, V. K. J. Org. Chem. 1973, 38, 2226.
(28) The position of the hydroxyl group of the product was determined
by a proton decoupling experiment. Also see: Behrens, C. H.; Sharpless,
K. B. J. Org. Chem. 1985, 50, 5696.
(29) Jung, S.-H.; Jeong, J.-H.; Miller, P.; Wong, C.-H. J. Org. Chem.
1994, 59, 7182.

7656 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Qiao et al.
Figure 4. Structures of aza sugars 1 and 2 and possible mode of inhibition caused by 2 (right). In the presence of 0.03 mM GDP, the concentration
of 2 necessary to achieve 50% inhibition of FucT V is 0.031 mM and that for 1 is 1.54 mM.
originally introduced by us31 and Schmidt32 with dibenzyl and
diethyl glycosyl phosphite employed as the glycosyl donors,
respectively. The bicyclic glycosyl phosphite 12 was obtained
easily by condensation of commercially available N,N-diethyl-
Figure 5. R-1,3-Fucosyltransferase V has an ordered, sequential
mechanism as determined by product inhibition patterns. GDP-fucose
is the donor sugar, N-acetyllactosamine (LacNAc) is the acceptor sugar,
and Lewis x (Lex) is the trisaccharide product.
1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine (10) with
Scheme 1a
2,3,4,5-tetra-O-acetyl-D-galactopyranose33 in the presence of 1H-
tetrazole (92%) (Scheme 3).
Compound 11 was chosen as the glycosyl acceptor and
was prepared by a standard reaction sequence.34 The 3-allyl
group not only activates the 4-OH for glycosylation but it
also can be manipulated further to allow the attachment of
the aza sugar moiety. Glycosylation of acceptor 11 with
glycosyl phosphite 12 catalyzed by TMSOTf (0.3 equiv) at
room temperature produced disaccharide 13 (38%); reactions
with the corresponding trichloroacetimidate as donor substrate
gave slightly lower yields (30%). Ozonolysis of the double
bond followed by reduction of the intermediate aldehyde
produced alcohol 14 (69%). One-pot triflate formation of
alcohol 14 and N-alkylation30a of aza sugar 9 provided the tar-
get skeleton 15 (63%). Although it appeared that the direct
reductive amination of the aldehyde intermediate obtained
by ozonolysis with aza sugar 1 could give the precursor to 2,
this approach proved to be problematic under a variety of
reaction conditions. Finally, saponification of the esters fol-
lowed by hydrogenolysis of the benzyl groups provided
compound 2 (91%).
a Reagents and conditions: (a) Ni(OAc)4‚4H2O, NaBH4, NH2-
(CH2)2NH2, then H2, 1 atm, 81%; (b) m-CPBA, NaHCO3, CH2Cl2, 71%;
(c) NaN3, NH4Cl, EtOH:H2O, 90 °C, 44%; (d) 0.1 N HCl, 50 °C, 5 h;
(e) (i) DHAP, FDP aldolase, pH 6.7, 25 °C; (ii) acid phosphatase, pH
4.7, 37 °C, 66%; (f) Pd/C, H2, 50 psi, 94%.
Scheme 2a
(30) (a) van den Broek, L. A. G. M.; Vermaas, D. J.; Heskamp, B. M.;
van Boeckel, C. A. A.; Tan, M. C. A. A.; Bolscher, J. G. M.; Ploegh, H.
L.; van Kemenade, F. J.; de Goede, R. E. Y.; Miedema, F. Recl. TraV.
Chim. Pays-Bas 1993, 112, 82. (b) Bernotas, R. C.; Cube, R. V. Synth.
Commun. 1990, 20, 1209.
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115, 2260.
(34) Compound 11 was prepared from N-acetyl-D-glucosamine by
glycosylation, benzylidene formation, allylation, and regioselective opening
of the benzylidene ring. (a) For glycosylation see: Lemieux, R. U.; Bundle,
D. R.; Baker, D. A. J. Am. Chem. Soc. 1975, 97, 4076. (b) For benzylidene
formation see: Evans, M. E. Carbohydr. Res. 1972, 21, 473. (c) For
allylation see: Durette, P. L.; Meitzner, E. P. Carbohydr. Res. 1981, 89,
279. (d) For reductive benzylidene ring opening see: Garegg, P. J.; Hultberg,
H. Carbohydr. Res. 1981, 93, C10.
a Reagents and conditions: (a) NaH, BnBr, -40 to 25 °C, 53%; (b)
Pd(OH)2/C (Pearlman’s catalyst), H2, 55 psi, 96%.
cis-relationship between H 4-5. Selective N-debenzylation30
of 8 by hydrogenolysis catalyzed by Pd(OH)2 on carbon
(Pearlman’s catalyst) provided free amine 9 ready for coupling
(51%, two steps).
Preparation of the protected LacNAc involved a glycosylation
with a novel bicyclic glycosyl phosphite 12. This convenient
and improved phosphite methodology is different from the ones

Inhibition of R-1,3-Fucosyltransferase V
J. Am. Chem. Soc., Vol. 118, No. 33, 1996 7657
Scheme 3a
a Reagents and conditions: (a) 10, 1H-tetrazole, THF, 25 °C, 92%; (b) 11, TMSOTf (0.3 equiv) CH2Cl2, 25 °C, 38%; (c) O3, CH2Cl2:MeOH,
then Me2S, -78 to 25 °C, then NaBH4, EtOH, 25 °C, 69%; (d) diisopropylethylamine, Tf2O, CH2Cl2, -20 °C, then 9, -20 to 25 °C, 63%; (e) (i)
NaOMe, MeOH; (ii) Pd(OH)2/C, (Degussa type), MeOH/AcOH, H2, 1 atm, 91%.
Table 1.
compound
LacNAc
GDP-fucose
Km, mM
Ki, mM (IC50)
35
0.009
Lewis x
GDP
52.3 (84.0)
0.0290 (0.067)
2.40 (5.7)
azatrisaccharide 2
azatrisaccharide 2a
homofuconojirimycin 1
homofuconojirimycin 1a
deoxyfuconojirimycin 16
deoxyfuconojirmycina 16
aza sugar 17
-
(0.031)
32.9 (71.5)
(1.54)
45.3 (73.1)
Figure 6. Inhibition of R-1,3-fucosyltransferase by 2. (A) Inhibition
with respect to the donor sugar, GDP-fucose (at 24 mM LacNAc). A
competitive pattern is observed. Nonlinear regression analysis of the
data with the Compo program reveals a Ki ) 2.7 mM. Concentrations
of inhibitor 2 were 0 (9), 4 (2), and 8 mM (b). (B) Inhibition with
respect to the acceptor sugar, LacNAc (at 0.05 mM GDP-fucose). Mixed
inhibition is observed resulting in a Ki ) 2.4 mM. Inhibitor concentra-
tions were 0 ([), 2 (9), 4 (2), and 8 mM (b).
-
-
-
(3.55)
(80)
a IC50 with all assays containing 0.030 mM GDP.
resulted in 80% inhibition in the presence of 24 mM LacNAc,
while individually only 15% and 29% inhibitions were observed,
respectively.
Inhibition Analysis. Inhibition studies with FucT V were
conducted with purified enzyme. Double reciprocal analysis
demonstrated that compound 2 had a competitive inhibition
pattern with respect to GDP-fucose (Figure 6a), and nonlinear
regression analysis22 of the data yielded a Ki ) 2.7 mM. The
mode of inhibition of 2 with respect to LacNAc was linear
mixed (Figure 6b) and the Ki was determined to be 2.4 mM.
â-L-Homofuconojirimycin (1) was determined to have a Ki of
32.9 mM by Dixon analysis.35 The Km of LacNAc has been
reported to be 35 mM.9 Therefore, inhibitor 2 is an order of
magnitude more potent than either of its components 1 and
LacNAc.
Synergy of aza sugars in combination with GDP was
evaluated for the inhibition of FucT V. Synergy is defined as
an interaction between the two inhibitors on the enzyme such
that the presence of one inhibitor decreases the dissociation
constant of the other (and vice versa). It would seem reasonable
to believe that both the fucose-type aza sugar 1 and GDP bind
to FucT V at the GDP-fucose site in the enzyme active site. At
their Ki levels, individually 1 and GDP caused 17% and 25%
inhibition in the presence of 24 mM LacNAc, respectively;
combination of 1 and GDP resulted in 79% inhibition of the
enzyme. Evaluation of the synergistic effect of 2 and GDP
demonstrated that combination of 2 and GDP at their Ki levels
Apparent IC50 values were determined for 1, 2, and deoxy-
fuconojirimycin (16) in the presence of GDP. The percent of
inhibition of FucT V by the azasaccharides in the presence of
0.030 mM GDP, 5 mM LacNAc, and 0.025 mM GDP-fucose
was determined relatiVe to a solution that contained 0.030 mM
GDP, 5 mM LacNAc, and 0.025 mM GDP-fucose. The
absolute amount of inhibition of FucT V at the apparent IC50
was greater than 50% because GDP is an inhibitor, but was
factored out by the experimental design. The IC50 values of 1,
2, 16 were determined to be 71.5, 5.7, and 73.1 mM,
respectively. In the presence of 0.030 mM GDP, the apparent
IC50 values of 1, 2, and 16 were 1.54, 0.031, and 3.55 mM,
respectively. The synergism of GDP, aza sugar and acceptor
sugar was responsible for a 22-77-fold decrease in the
concentration of aza sugar necessary to cause 50% inhibition
(IC50) (Table 1).
The kinetic proof of synergistic inhibition is illustrated in
Scheme 4. Since GDP and fucose-type aza sugars are competi-
tive inhibitors with respect to GDP-fucose, they both bind only
free enzyme; this is analogous to the single substrate system
described by Yonetani and Theorell.36 GDP is a product
inhibitor of FucT V and the kinetics of such a system has been

7658 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Qiao et al.
Scheme 4. Kinetic Mechanism for FucT V Including Synergistic Inhibition by the Combination of GDP and an Aza Sugar (I)a
a FucT V has an ordered, sequential BiBi mechanism.
described (eq 1).25 Fucose-type aza sugars should interact with
FucT V in a mode similar to that of GDP. Therefore, we can
extend the rate equation for GDP inhibition (eq 1) to an equation
for synergistic inhibition (eq 3) by including terms for aza sugar
inhibition ([aza sugar]/Ki,aza sugar) and for the formation of the
GDP-aza sugar-enzyme complex ([aza sugar][GDP]/RKi,aza
-
sugarKi,GDP). The data obtained can be fitted to the following
equation:
V ) Vmax[GDP-fucose]/[Km,GDPfucI*{1 +
Ki,GDP-fucKm,LacNAc/[Km,GDP-fuc[LacNAc])} +
[GDP-fuc](1 + Km,LacNAc/[LacNAc])]
Figure 7. Multiple inhibition studies of FucT V. (A) the Yonetani-
Theorell plot reveals nonexclusive binding by the pattern of intersecting
lines. The intersection point (RKi) reveals that R < 1, indicative of
synergistic inhibition. LacNAc (24 mM) and GDP-fucose (0.05 mM)
were held at fixed concentrations. GDP concentrations were as
follows: 0.0 mM (b), 0.05 mM ([), 0.10 mM (+), 0.025 mM (2),
and 1/Vmax (9). (B) Role of the acceptor sugar, LacNAc, in the
synergistic inhibition of Fuc-T V by the combination of the 2 and GDP.
The acceptor sugar had a modest effect on the inhibition of single
inhibitors but had a substantial effect on the inhibition of a combination
of 2 and GDP. GDP-fucose was held constant at 0.05 mM. No inhibitors
(b), 0.03 mM GDP ([), 2.8 mM 2 (9) and 0.03 mM GDP + 2.8 mM
2 (2).
[GDP] [aza sugar] [GDP][aza sugar]
I* ) 1 +
+
+
(3)
Ki,GDP
Ki,aza sugar
RKi,GDPKi,aza sugar
The variable R is the interaction constant of the two inhibitors,
aza sugar and GDP. If R > 1, the binding of one inhibitor
hinders the binding of the other (antisynergy); if R ) 1, the
binding of one inhibitor has no effect on the binding of the
other; and if R < 1, the inhibitors bind synergistically. Equation
3 can be rearranged into a linear expression in the form of Dixon
plots. At constant substrate concentration, plots of velocity
versus the inhibitor concentration, [I], at different fixed con-
centrations of the other inhibitor, X, give lines that intersect at
a y-value less than 1/Vmax for R > 1 (antisynergy), lines that
intersect at a y-value equal to 1/Vmax for R ) 1, or lines that
intersect at a y-value greater than 1/Vmax for R < 1 (synergy).
Yonetani-Theorell plots for the combination of GDP and 2
showed a pattern of nonexclusive, synergistic inhibition; a family
of lines intersecting at a y-value greater than 1/Vmax and [I] )
-RKi (Figure 7A). The interaction constant R was determined
to be 0.18, showing that the presence of one inhibitor increases
the affinity of the other by a factor of 6-fold (1/R); and the
-RKi was determined to be 0.45 mM.
The role of the acceptor sugar in the synergistic inhibition
of FucT V was investigated by monitoring FucT V activity as
a function of LacNAc concentration at fixed GDP-fucose, GDP,
and aza sugar concentrations. The concentrations of LacNAc
used in the study were from 5 to 40 mM. The effect of the
individual inhibitors on FucT V as a function of LacNAc was
modest, which is in agreement with the inhibition studies
at 24 mM LacNAc. The combination of the aza sugars with
GDP was subject to a substantial acceptor sugar effect. The
combination of 2.8 mM 2 and 0.030 mM GDP produced a
linear effect in the double reciprocal plot despite the fact that 2
contains a LacNAc moiety (Figure 7B). The apparent Km of
LacNAc under the experimental conditions is 35 mM and the
maximum LacNAc concentration (40 mM) was not saturating.
The mono aza sugars were subject to an acceptor sugar effect.
Both â-L-homofuconojirimycin (1) and the 5-membered fucose-
type aza sugar 178 in combination with 0.05 mM GDP produced
a substantial, linear acceptor sugar effect in the double reciprocal
plot (Figure 8). These studies indicate a role for the acceptor
sugar in the synergistic inhibition of FucT V through a
quaternary complex of FucT V, acceptor sugar, GDP, and aza
sugar, and suggest that certain aza sugars which inhibit
glycosidases may also inhibit glycosyltransferases in ViVo.
Mechanism. The synergistic inhibition of fucosyltransferase
displayed by aza sugars and GDP indicates the interaction of
the positively charged aza sugars with both the negatively
charged GDP and the bound acceptor sugar in the active site,
which may mimic the transition state of the fucosyltransfer
reaction as shown in Figure 2. The increased inhibitory potency
of 2 over simple aza sugars such as 1 provides further indication.
The synergism of GDP and 2 was responsible for an additional
77-fold enhancement in inhibitory potency. Since transition
state analogs are expected to bind tightly to the enzymes,37 the
moderate inhibition of 2 may reflect the nonideal length of the
linker moiety, which resulting in the less than perfect orientation
of the LacNAc or 1 in their respective binding pockets. Initial
(35) Dixon, M.; Webb, E. C. In Enzymes, 3rd ed.; Academic Press: New
York, 1979; p 350-351
(36) Yonetani, T.; Theorell, H. Arch. Biochem. Biophys. 1964, 106, 243.
(37) (a) Pauling, L. Chem. Eng. News 1946, 24, 1375. (b) Pauling, L.
Am. Scientist 1948, 36, 51.

Inhibition of R-1,3-Fucosyltransferase V
J. Am. Chem. Soc., Vol. 118, No. 33, 1996 7659
Experimental Section
General Methods. All processes involving air or moisture sensitive
reactants were done under an atmosphere of dry argon using oven-
dried glassware. Reagents and solvents were reagent grade and used
as supplied unless otherwise mentioned. Solvent evaporation was
performed under reduced pressure below 40 °C using a Bu¨chi rotary
evaporator, followed by evacuation (<0.1 Torr) to constant sample
weight. High resolution mass spectra (HRMS) were recorded on a
VG ZAB-ZSE instrument under fast atom bombardment (FAB)
conditions. 1H NMR spectra were obtained at 400 MHz and 13C NMR
at 100 MHz on a Bruker AM-400 instrument. 1H NMR chemical shifts
are reported in parts per million (ppm) downfield relative to tetra-
methylsilane (TMS) using the solvent resonance as the reference:
CDCl3 δ 7.26, HDO δ 4.60; 13C NMR chemical shifts are reported
relative to CDCl3 δ 77.0. Purified soluble R-1,3-Fuc-T V was a
generous gift from the Cytel Corporation (La Jolla, CA). N-Acetyl-
lactosamine, guanosine 5′-diphosphate, guanosine-5′-diphospho-â-
fucose, adenosine 5′-triphosphate, and FDP aldolase were purchased
from Sigma. Guanosine-5′-diphospho-L-[U-14C]fucose was purchased
from Amersham Life Science. ScintiVerseI scintillation cocktail was
purchased from Fisher. Scintillation counting was performed on a
Beckman LS 3801 instrument.
cis-2-Buten-l-al Diethyl Acetal (4). Sodium borohydride (147 mg,
3.9 mmol) in EtOH (4 mL) was added dropwise to a solution of nickel
acetate tetrahydrate (0.98 g, 3.9 mmol) in EtOH (40 mL). The resulting
mixture was stirred for a an additional 10 min and then ethylene diamine
(0.52 mL, 7.8 mmol) and butyn-1-al diethyl acetal (5.0 g, 35.1 mmol)
were added. The mixture was stirred under a H2 atmosphere for 6 h,
and then filtered though a pad of Celite. To the filtrate was added
H2O and it was extracted with ether (60 mL × 3). The combined
extracts were washed with brine, dried (MgSO4), and concentrated
below 20 °C to give olefin 4 (4.1 g, 81%) as a colorless and volatile
oil: 1H NMR (CDCl3, 400 MHz) δ 5.75-5.65 (m, 1 H, H-2), 5.50-
5.42 (m, 1 H, H-3), 5.20 (d, J ) 7.0 Hz, 1 H, H-1), 3.68-3.58 (m, 2
H, OCH2CH3), 3.50-3.42 (m, 2 H, OCH2CH3), 1.70 (dd, J ) 7.0, 1.5
Hz, 3 H, CH3), 1.22 (t, J ) 7.0 Hz, 6 H, 2 × OCH2CH3); 13C NMR
(CDCl3, 100 MHz) δ 128.99, 128.16, 97.47, 60.49, 15.29, 13.61.
cis-2,3-Epoxybutyraldehyde Diethyl Acetal (5). A mixture of
olefin 4 (211 mg, 1.46 mmol), m-CPBA (503 mg of a solid of 57-
86% purity, 2 mmol), and NaHCO3 (132 mg, 1.57 mmol) in CH2Cl2
(25 mL) was stirred at room temperature for 12 h. Saturated Na2SO3
solution (10 mL) was then added slowly and the resulting mixture was
stirred for 20 min. The organic layer was separated, washed with
saturated NaHCO3, H2O, and brine, and dried (MgSO4). The solvent
was removed in Vacuo below 20 °C to give epoxide 5 (165 mg, 71%)
as a volatile oil: 1H NMR (CDCl3, 400 MHz) δ 4.30 (d, J ) 6.5 Hz,
1 H, H-1), 3.80-3.52 (m, 4 H, 2 × OCH2CH3), 3.13-3.02 (m, 2 H,
H-2, H-3), 1.25 (t, J ) 7.0 Hz, 3 H, OCH2CH3), 1.22 (t, J ) 7.0 Hz,
3 H, OCH2CH3); 13C NMR (CDCl3, 100 MHz) δ 100.33, 62.08, 56.37,
51.87, 15.33, 15.25.
Figure 8. Role of the acceptor sugar (LacNAc) in the synergistic
inhibition of FucT V with the combination of aza monosaccharides
and GDP. The effect of the individual inhibitors on FucT V was
modest. The combination of the aza sugars with GDP was subject to
a substantial acceptor sugar effect. GDP-fucose and GDP were both
held constant at 0.05 mM. The concentrations are as follows: 33 mM
1 + 0.050 mM GDP (9), 17 + 0.030 mM GDP (b). (Inset) The
concentrations of the inhibitors are as follows: no inhibitors (9), 33
mM 1 (×), 80 mM 17 (b), 0.050 mM GDP (2).
computer modeling studies reveal that 2 is not an ideal Lex
mimetic due to the length of the linker; therefore, variation of
the linker could lead to better inhibitors. Acceptor specificity
studies have demonstrated that FucT V has unusually high Km
values for all substrates studied (e.g. LacNAc, Km ) 35
mM);7a,8,9 therefore, it is likely that more potent inhibition and
profound synergistic inhibition may occur with an inhibitor
comprised of a more specific substrate than LacNAc, as
illustrated in other studies.38-42
In our recent study on the specificity and mechanism of FucT
V,43 a pH-rate profile revealed one catalytically relevant residue
with a pKa ) 4.1. This result is consistent with a carboxylic,
general-base-catalyzed mechanism. FucT V was shown to be
subject to a solvent isotope effect (DV/K ) 2.1, DV ) 2.9) and
a proton inventory study that revealed that one exchangeable
proton was involved in the catalytic mechanism.43 Detailed
kinetic studies on FucT V yielded the following parameters:43
kcat ) 40 min-1, Ki,GDP-fuc ) 0.0062 mM, Km,GDP-fuc ) 0.060
mM, Km,LacNAc ) 8.8 mM. The enzyme proficiency of FucT
V, (kcat/Ki,GDP-fucKm,LacNAc)/knon, is estimated to be 1.2 × 1010
M-1 and the transition-state affinity is therefore 8.6 × 10-11
M.43 This places FucT V at the lower end of the range of
transition state affinities (5.3 × 108 to 2.0 × 1023 M-1),44 which
may contribute to the difficulty in the development of potent
fucosyltransferase inhibitors. However, given the low affinity
of FucT V for its substrate (Km ) 35 mM for LacNAc), the
designed azatrisaccharide in the presence of GDP represents
the most potent transition-state analog inhibitor (IC50 ) 31 µM)
reported so far.
threo-3-Azido-2-hydroxybutyraldehyde Diethyl Acetal (6).
A
mixture of epoxide 5 (2.4 g, 15 mmol), sodium azide (4.9 g, 75 mmol),
and ammonium chloride (4.0 g, 75 mmol) in EtOH (135 mL) and H2O
(15 mL) was heated at 90 °C for 20 h. The reaction mixture was diluted
with CH2Cl2 (100 mL) and filtered. The filtrate was concentrated in
Vacuo to about 50 mL and then extracted with CH2Cl2 (20 mL × 3).
The combined extracts were washed with brine, dried (MgSO4), and
concentrated in Vacuo. Filtration through a short silica gel columm
(EtOAc:hexane 1:10 to 1:5) and concentration gave the title compound
(1.35 g, 44%) as an yellowish volatile oil: 1H NMR (CDCl3, 400 MHz)
δ 4.48 (d, J ) 6.5 Hz, 1 H, H-1), 3.82-3.69 (m, 2 H, OCH2CH3),
3.60-3.48 (m, 3 H, H-3, OCH2CH3), 3.41 (dd, J ) 6.5, 2.0 Hz, H-2),
1.41 (d, J ) 7.0 Hz, 3 H, CH3), 1.20 (t, J ) 7.0 Hz, 3 H, OCH2CH3),
1.16 (t, J ) 7.0 Hz, 3 H, OCH2CH3); 13C NMR (CDCl3, 100 MHz) δ
102.90, 74.75, 64.46, 63.48, 56.43, 15.78, 15.41, 15.34.
(38) Gruys, K. J.; Walker, M. C.; Sikorski, J. A. Biochemistry 1992, 31,
5535.
(39) Seidel, H. M.; Knowles, J. R. Biochemistry, 1994, 33, 5641.
(40) Fletcher, R. S.; Arion, D.; Borkow, G.; Wainberg, M. A.; Dmit-
rienko, G. I.; Parniak, M. A. Biochemistry 1995, 34, 10106.
(41) England, P.; Herve, G. Biochemistry 1994, 33, 3913.
(42) (a) Pfuetzner, R. A.; Chan, W. W.-C. J. Biol. Chem. 1988, 263,
4056. (b) Xue, Y.; Huang, S.; Liang, J.-Y.; Zhao, Y.; Lipscomb, W. N.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12482. (c) Janc, J. W.; O’leary, M.
H.; Cleland, W. W. Biochemistry, 1992, 31, 6421.
A small amount of the title compound was acetylated with acetic
anhydride and pyridine to give the corresponding acetate: 1H-NMR
(CDCl3, 250 MHz) δ 4.92 (dd, J ) 7.0, 2.0 Hz, 1 H, H-2), 4.59 (d, J
) 7.0 Hz, 1 H, H-1), 3.80-3.50 (m, 5 H, 2 × OCH2CH3, H-3), 2.10
(s, 3 H, COCH3), 1.26-1.10 (m, 9 H, 3 × CH3).
6-Azido-6,7-dideoxy-L-galacto-heptulose (7). A mixture of threo-
3-azido-2-hydroxybutyraldehyde diethyl acetal (1.14 g, 5.63 mmol) in
(43) Murray, B. W.; Takayama, S.; Wong, C.-H. Biochemistry, submitted.
(44) Radzicka, A.; Wolfenden, R. Science 1995, 267, 90-93.

7660 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Qiao et al.
HCl (0.1 N, 30 mL) was heated at 50 °C for 5 h. The reaction mixture
was cooled to room temperature and the pH was adjusted to 7.0 with
5.0 N NaOH. Dihydroxyacetone phosphate29 (5.62 mL of a 0.50 M
solution, 2.81 mmol) was added and the pH was adjusted to 6.7 with
1 N NaOH. FDP aldolase (1134 units) was added, and the reaction
mixture was stirred at room temperature. After 12 h, the pH of the
reaction mixture (6.2) was adjusted to 6.7 with 1 N NaOH and then
the reaction mixture was stirred for an additional 28 h. The pH of the
reaction mixture was adjusted to 4.7 with 1.0 N HCl, and acid
phosphatase (750 units) was added. The mixture was incubated at 37
°C for 22 h. The reaction mixture was neutralized to pH ) 7.0 with
1.0 N NaOH and concentrated in Vacuo below 30 °C. The residue
obtained was extracted with MeOH (15 mL × 4), and the combined
extracts were concentrated. Flash chromatography (silica, CH2Cl2:CH3-
OH, 15:1) of the residue gave 7 (407 mg, 66%), as a yellowish oil,
and the other diastereomer (130 mg, 21%).
86.36, 77.74, 76.62, 75.27, 74.27, 73.16, 72.29, 69.40, 59.58, 53.29,
18.10; HRMS (LSIMS+) m/z calcd for C35H39NO4 + H+ 538.2957,
found 538.2945.
Propyl 2-Acetamido-3-O-allyl-6-O-benzyl-2-deoxy-â-D-glucopy-
ranoside (11). The title compound was prepared from N-acetyl-D-
glucosamine by a reaction sequence involving glycosylation, benzylide-
nation, allylation, and reductive ring opening of the benzylidene ring.34
1H NMR (CDCl3, 400 MHz) δ 7.40-7.20 (m, 5 H, ArH), 5.95-5.85
(dddd, J ) 17.2, 10.3, 5.8, 5.6 Hz, 1 H, OCH2CH)CH2), 5.84-5.82
(br s, 1 H, NH), 5.26 (d, J ) 17.2 Hz, 1 H, OCH2CHdCHH), 5.17 (d,
J ) 10.3 Hz, 1 H, OCH2CHdCHH), 4.90 (d, J ) 8.2 Hz, 1 H, H-1),
4.58 (ABq, J ) 12.0 Hz, 2 H, OCH2Ph), 4.25 (dd, J ) 12.6, 5.6 Hz,
1
H, OCHHCHdCH2), 4.18 (dd, J ) 12.6, 5.8 Hz, 1 H,
OCHHCHdCH2), 3.95 (dd, J ) 10.2, 8.3 Hz, 1 H, H-3), 3.82-3.72
(m, 3 H, H-6a, OCH2CH2CH3), 3.58 (br m, 2 H, H-4, H-6b), 3.41 (ddd,
J ) 9.6, 6.9, 6.9 Hz, 1 H, H-5), 3.15 (ddd, J ) 10.1, 8.04, 8.04 Hz, 1
H, H-2), 3.10 (br s, 1 H, OH), 1.98 (s, 3 H, CH3CO), 1.58 (dq, J )
7.4, 7.2 Hz, 2 H, OCH2CH2CH3), 0.88 (t, J ) 7.4 Hz, 3 H, OCH2-
CH2CH3); 13C NMR (CDCl3, 100 MHz) δ 170.51, 137.72, 134.99,
128.44, 127.80, 127.74, 117.21, 99.56, 80.13, 73.66, 73.56, 73.35, 73.26,
71.35, 70.65, 57.58, 23.62, 22.74, 10.34; HRMS (LSIMS+) m/z calcd
for C21H31O6N + Na+ 416.2049, found 416.2032.
For 7: 1H NMR (D2O, 400 MHz) δ 4.20-4.10 (m, 2 H), 3.70-
3.50 (m, 4 H), 1.30 (m, 3 H); 13C NMR (CDCl3, 100 MHz) δ 83.38,
78.92, 77.91, 66.18, 58.88, 17.42; HRMS (LSIMS+) m/z calcd for
C7H13N3O5 + Na+ 242.0753, found 242.0748.
2,6,7-Trideoxy-2,6-imino-L-glycero-D-manno-heptitol (â-L-Homo-
fuconojirimycin) (1). A solution of azidoketose 7 (410 mg, 1.87 mmol)
in methanol (30 mL) was hydrogenated at 50 psi in the presence of
10% Pd/C (40 mg) for 24 h. The catalyst was removed by filtration
through a pad of Celite. Concentration of the filtrate in Vacuo gave
aza sugar 1 (310 mg, 94%) as a foam: 1H NMR (D2O, 400 MHz) δ
3.80-3.70 (m, 3 H, H-5, H-1a, H-1b), 3.56-3.54 (m, 2 H, H-3, H-4),
2.85 (dd, J ) 7.0, 7.0 Hz, 1 H, H-6), 2.52-2.48 (m, 1 H, H-2), 1.10
(d, J ) 7.0 Hz, 3 H, CH3); 13C NMR (D2O, 100 MHz) δ 77.42, 74.81,
70.25, 62.95, 62.27, 54.63, 18.61; HRMS (LSIMS+) m/z calcd for
C7H15NO4 + H+ 178.1079, found 178.1083.
1,3,4,5-Tetra-O-benzyl-2,6-(N-benzylimino)-2,6,7-trideoxy-L-glyc-
ero-D-manno-heptitol (8). At -50 °C, NaH (164 mg of a 60%
dispersion in oil, 4.11 mmol, washed with dry hexane before use)
was added to a solution of aza sugar 1 (112 mg, 0.633 mmol) in
dry DMF (2.5 mL). After being stirred for 5 min, the reaction mix-
ture was allowed to warm to 0 °C, and benzyl bromide (0.70 mL,
5.88 mmol) was added. After being stirred at 0 °C for 5 h and
room temperature for 4 h, the reaction mixture was diluted with
EtOAc (20 mL) and washed with H2O and brine, dried (MgSO4), and
concentrated. Purification of the residue by flash chromatography
(silica, hexane:EtOAc, gradient, 30:1 to 10:1) gave compound 8
(209 mg, 53%) as a colorless oil: 1H NMR (CDCl3, 400 MHz) δ
7.40-7.15 (m, 25 H, ArH), 4.92 (d, J ) 11.8 Hz, 1 H, OCHHPh),
4.80 (d, J ) 11.0 Hz, 1 H, OCHHPh), 4.75 (ABq, J ) 11.8 Hz, 2 H,
OCH2Ph), 4.65 (d, J ) 11.8 Hz, 1 H, OCHHPh), 4.55 (d, J ) 11.0 Hz,
1 H, OCHHPh), 4.20 (ABq, J ) 12.0 Hz, 2 H, OCH2Ph), 4.15 (d,
J ) 16.6 Hz, 1 H, NCHHPh), 4.05 (dd, J ) 8.0, 8.0 Hz, 1 H, H-3),
3.98 (d, J ) 16.5 Hz, 1 H, NCHHPh), 3.80 (t, J ) 2.8 Hz, 1 H.
H-1a), 3.70 (m, 2 H, H-5, H-1b), 3.61 (dd, J ) 8.0, 2.9 Hz, 1 H, H-4),
3.00-2.90 (m, 2 H, H-2, H-6), 1.08 (d, 3 H, J ) 7.0 Hz, 3 H,
CH3); 13C NMR (CDCl3, 100 MHz) δ 141.97, 139.23, 138.84, 138.77,
138.40, 128.34, 128.28, 128.24, 128.21, 128.15, 127.92, 127.86, 127.76,
127.73, 127.44, 127.35, 127.29, 126.15, 84.29, 78.44, 77.35, 77.03,
76.71, 75.40, 74.13, 73.84, 72.87, 72.55, 68.75, 64.04, 56.98, 52.55,
16.65; HRMS (LSIMS+) m/z calcd for C42H45NO4 + Cs+ 760.2403,
found 760.2425.
3-(2,3,4,6-Tetra-O-acetyl-D-galactopyranosyl)-1,5-dihydro-2,4,3-
benzodioxaphosphepin (12). At room temperature, N,N-diethyl-1,5-
dihydro-2,4,3-benzodioxaphosphepin-3-amine (10) (370 µL, 1.72 mmol)
was added to a solution of D-galactose tetraacetate (300 mg, 0.862
mmol) and 1H-tetrazole (241 mg, 3.45 mmol) in dry THF (3.0 mL)
under an argon atmosphere. After the reaction mixture was stirred for
2.5 h, CH2Cl2 (20 mL) and saturated NaHCO3 (20 mL) were added.
The organic layer was separated, washed with saturated NaHCO3, dried
(MgSO4), and concentrated. Purification of the residue by flash
chromatography (silica, CH2Cl2:MeOH:Et3N, 125:1:0.25) gave phos-
phite 12 (397 mg, 92%) as a mixture of R,â-anomers as well as fractions
containing pure R-anomer.
For R-anomer: 1H NMR (CDCl3, 400 MHz) δ 7.25-7.18 (m, 4 H,
ArH), 5.81-5.76 (m, 2 H, H-1, OCHHAr), 5.70 (dd, J ) 13.4, 10.8
Hz, 1 H, OCHHAr), 5.49 (br d, J ) 2.5 Hz, 1 H, H-4), 5.40 (dd, J )
10.8, 3.3 Hz, 1 H, H-3), 5.22 (dd, J ) 10.8, 3.5 Hz, 1 H, H-2), 4.65
(dd, J ) 13.4, 9.7 Hz, 1 H, OCHHAr), 4.57 (dd, J ) 13.4, 9.9 Hz, 1
H, OCHHAr), 4.48 (br t, J ) 6.4 Hz, 1 H, H-5), 4.14 (dd, J ) 11.3,
6.4 Hz, 1 H, H-6a), 4.08 (dd, J ) 11.3, 6.9 Hz, 1 H, H-6b), 2.13 (s, 3
H, CH3CO), 2.06 (s, 3 H, CH3CO), 1.99 (s, 3 H, CH3CO), 1.98 (s, 3
H, CH3CO); 13C NMR (CDCl3, 100 MHz) δ 170.38, 170.17, 170.14,
170.06, 137.74 (d, J ) 4.0 Hz), 128.40 (d, J ) 2.0 Hz), 128.20, 91.48
(d, J ) 21.6 Hz), 67.94 (d, J ) 4.0 Hz), 67.74, 67.50, 67.41, 64.89 (d,
J ) 150 Hz), 61.52, 20.67, 20.62; 31P NMR (CDCl3, 162 MHz) δ
131.98; HRMS (LSIMS+) m/z calcd for C22H27O12P + Cs+ 647.0294,
found 647.0271.
Propyl 2-N-Acetamido-3-O-allyl-6-O-benzyl-2-O-deoxy-4-O-(2,3,4,6-
tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopyranoside (13). At
room temperature, TMSOTf (22.0 µL, 0.112 mmol) was added dropwise
to a solution of alcohol 11 (147 mg, 0.375 mmol) and phosphite 12
(224 mg, 0.45 mmol) in dry CH2Cl2 (1.2 mL) over a period of 1.5 h.
The reaction mixture was stirred at room temperature for 18 h and
CH2Cl2 (20 mL) was then added. The resulting solution was washed
with saturated NaHCO3, dried (MgSO4), and concentrated. Purification
by flash chromatography (silica, CH2Cl2:MeOH, gradient, 100:1 to 80:
1) gave disaccharide 13 (103 mg, 38%) as a colorless oil: 1H NMR
(CDCl3, 400 MHz) δ 7.40-7.25 (m, 5 H, ArH), 6.11 (d, J ) 8.4 Hz,
1 H, NH), 5.85 (dddd, J ) 17.2, 10.3, 5.8, 5.6 Hz, 1 H, OCH2CH)CH2),
5.32 (d, J ) 2.8 Hz, 1 H, H-4′), 5.22 (dd, J ) 17.2 , 1.6 Hz, 1 H,
OCH2CH)CHH), 5.15-5.05 (M, 2 H, H-2′, OCH2CH)CHH), 4.90
(dd, J ) 10.5, 3.4 Hz, 1 H, H-3′), 4.72 (d, J ) 5.7 Hz, 1 H, H-1′), 4.65
(d, J ) 12.0 Hz, 1 H, OCHHPh), 4.50-4.46 (m, 2 H, H-1, OCHHPh),
4.20-4.05 (m, 4 H), 3.90-3.55 (m, 8 H), 3.35 (dt, J ) 9.4, 6.8 Hz, 1
H), 2.12 (s, 3 H, CH3CO), 2.02-1.96 (4 × s, 4 × COCH3), 1.55 (sextet,
J ) 7.0 Hz, OCH2CH2CH3), 0.87 (t, J ) 7.0 Hz, 3 H, OCH2CH2CH3);
13C NMR (CDCl3, 100 MHz) δ 170.29, 170.18, 170.01, 169.89, 137.98,
134.87, 128.48, 127.91, 116.58, 99.76, 99.73, 76.69, 75.28, 74.10, 73.50,
72.03, 70.99, 70.60, 70.56, 69.17, 68.85, 66.82, 60.93, 53.15, 23.38,
22.72, 20.83, 20.65, 20.55, 10.43; HRMS (LSIMS+) m/z calcd for
C35H49NO15 + Cs+ 856.2157, found 856.2183.
1,3,4,5-Tetra-O-benzyl-2,6-imino-2,6,7-trideoxy-L-glycero-D-manno-
heptitol (9). A mixture of compound 8 (209 mg, 0.333 mmol) and
20% Pd(OH)2/C (Pearlman’s catalyst, 108 mg) in EtOAc:EtOH (18:3
mL) was hydrogenated at 55 Psi for 16 h. Filtration and concentration
gave amine 9 (172 mg, 96%) as an oil: 1H NMR (CDCl3, 400 MHz)
δ 7.40-7.15 (m , 20 H, ArH), 5.05 (d, J ) 11.5 Hz, 1 H, OCHHPh),
4.89 (d, J ) 11.0 Hz, 1 H, OCHHPh), 4.73 (ABq, J ) 11.5 Hz, 2 H,
OCHHPh), 4.67 (d, J ) 11.5 Hz, 1 H, OCHHPh), 4.51 (d, J ) 11.5
Hz, 1 H, OCHHPh), 4.48 (d, J ) 11.0 Hz, 1 H, OCHHPh), 4.42 (d, J
) 11.5 Hz, 1 H, OCHHPh), 3.91 (dd, J ) 9.5, 9.5 Hz, 1 H, H-3),
3.71-3.68 (m, 2 H, H-5, H-1a), 3.61 (dd, J ) 9.0, 2.5 Hz, 1 H, H-1b),
3.54 (dd, J ) 9.5, 2.0 Hz, 1 H, H-4), 2.75-2.65 (m, 2 H, H-2, H-6),
1.09 (d, J ) 6.7 Hz, 3 H, CH3); 13C NMR (CDCl3, 100 MHz) δ 138.65,
128.40, 128.32, 128.16, 128.13, 127.82, 127.64, 127.55, 127.50, 127.35,

Inhibition of R-1,3-Fucosyltransferase V
J. Am. Chem. Soc., Vol. 118, No. 33, 1996 7661
Propyl 2-Acetamido-3-O-(2-hydroxyethyl)-6-O-benzyl-2-deoxy-
4-O-(2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-â-D-glucopyrano-
side (14). At -78 °C, ozone was bubbled through a solution of olefin
13 (325 mg, 0.449 mmol) in CH2Cl2 (20 mL) until a blue color
persisted. This solution was kept at -78 °C for an additional 5 min
and then purged with O2 to remove the excess O3. Methyl sulfide (0.42
mL) was added, and the resulting mixture was kept at -78 °C for 1 h
and room temperature for 1 h. The mixture was then concentrated in
Vacuo and the residue was dissolved in EtOH (3 mL). NaBH4 (8.2
mg, 0.216 mmol) in H2O (0.3 mL) was added to the solution and the
mixture was stirred at room temperature. After 20 min, 1 N HCl (0.3
mL) was added and the resulting mixture was stirred for an additional
5 min. Concentration in Vacuo and purification by flash chromatog-
raphy (silica, CH2Cl2:MeOH, 35:1) gave alcohol 14 (225 mg, 69%) as
a colorless oil: 1H NMR (CDCl3, 400 MHz) δ 7.40-7.25 (m, 5 H,
ArH), 6.05 (d, J ) 8.1 Hz, 1 H, NH), 5.20 (d, J ) 2.4 Hz, 1 H, H-4′),
5.10 (dd, J ) 10.4, 8.1 Hz, 1 H, H-2′), 4.84 (dd, J ) 10.4, 3.4 Hz, 1
H, H-3′), 4.79 (d, J ) 6.4 Hz, 1 H, H-1), 4.70 (d, J ) 12 Hz, 1 H,
OCHHPh), 4.48 (d, J ) 8.0 Hz, 1 H, H-1′), 4.46 (d, J ) 12.0 Hz, 1 H,
OCHHPh), 4.16 (dd, J ) 11.3, 6.4 Hz, 1 H, H-6′a), 4.08 (dd, J )
11.3, 7.0 Hz, 1 H, H-6′b), 3.90-3.34 (m, 13 H), 3.18 (br s, 1 H, OH),
2.13 (s, 3 H, CH3CO), 2.03 (s, 3 H, CH3CO), 1.99 (s, 6 H, 2 × CH3-
CO), 1.96 (s, 3 H, CH3CO), 1.56 (sextet, J ) 7.4 Hz, 2 H, OCH2CH2-
CH3), 0.87 (t, J ) 7.4 Hz, 3 H, CH3); 13C NMR (CDCl3, 100 MHz) δ
170.60, 170.30, 170.00, 169.44, 137.75, 128.56, 128.05, 128.00, 127.81,
99.81, 99.53, 77.66, 75.12, 74.11, 73.84, 73.55, 71.28, 70.80, 70.64,
69.10, 68.23, 66.72, 61.66, 60.79, 23.37, 22.64, 20.74, 20.64, 20.54,
10.36; HRMS (LSIMS+) m/z calcd for C34H49NO16 + Cs+ 860.2106
found 860.2126.
Propyl 2-Acetamido-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-â-D-ga-
lactopyranosyl)-6-O-benzyl-3-O-(2-(N-(1,3,4,5-tetra-O-benzyl-â-L-
homofuconojirimycinyl))ethyl)-r-D-glucopyranoside (15). A solution
of alcohol 14 (115 mg, 0.158 mmol) and diisopropylethylamine (0.2
mL, 1.14 mmol) in dry CH2Cl2 (2.0 mL) was cooled to -20 °C. Tf2O
(35 µL, 0.205 mmol) was added slowly, and the resulting mixture was
kept at -20 °C for 5 h. An additional portion of Tf2O (10 µL, 0.06
mml) was then added, and the reaction mixture was stirred at -20 °C
for an additional 20 min. Aza sugar 9 (56 mg, 0.104 mmol) in CH2-
Cl2 (0.25 mL) was added dropwise, and the resulting mixture was
allowed to warm to room temperature. After the reaction mixture was
stirred for 20 h, CH2Cl2 (10 mL), 0.5 N NaOH (2 mL), and saturated
NaHCO3 (10 mL) were added. The organic layer was separated and
washed with brine, dried (MgSO4), and concentrated. Purification of
the residue by flash chromatography gave 15 (81 mg, 63%) as a
colorless oil: 1H NMR (CDCl3, 400 MHz) δ 7.40-7.08 (m, 25 H,
ArH), 6.25 (d, J ) 3.1 Hz, 1 H), 5.05 (dd, J ) 10.2, 8.2 Hz, 1 H), 4.94
(d, J ) 8.2 Hz, 1 H), 4.81-4.40 (m, 12 H), 4.30-3.24 (m, 23 H), 2.30
(s, 3 H), 2.12 (s, 3 H), 2.02 (s, 3 H), 1.96 (s, 3 H), 1.95 (s, 3 H),
1.58-1.50 (m, 2 H), 1.27 (d, J ) 6.2 Hz, 3 H), 0.82 (t, J ) 7.4 Hz, 3
H); 13C NMR (CDCl3, 100 MHz) δ 170.32, 170.12, 168.66, 165.96,
137.83, 137.64, 137.27, 137.01, 128.62, 128.58, 128.47, 128.42, 128.37,
128.16, 128.12, 128.01, 127.95, 127.84, 127.67, 99.69, 99.51, 80.79,
77.48, 77.20, 76.12, 75.33, 74.86, 73.78, 73.56, 73.33, 73.04, 71.96,
71.69, 71.01, 70.45, 69.47, 69.05, 67.62, 67.33, 66.85, 63.78, 62.15,
61.59, 60.81, 29.68, 22.81, 20.67, 20.62, 20.57, 16.87, 14.00, 10.40;
HRMS (LSIMS+) m/z calcd for C69H86N2O19 + H+ 1347.5903, found
1347.5950.
(12 mg, 91%) as a glassy solid: 1H NMR (D2O, 400 MHz) δ 4.50-
4.20 (m, 4 H), 4.02-3.45 (m, 21 H), 3.20-3.05 (m, 2 H), 2.15 (s, 3
H), 1.65 (sextet, J ) 7.1 Hz, 2 H), 1.39 (d, J ) 6.7 Hz, 3 H), 0.90 (t,
J ) 7.4 Hz, 3 H); 13C NMR (D2O, 100 MHz) δ 175.91, 105.30, 101.25,
78.35, 77.85, 76.28, 75.47, 74.93, 73.62, 72.26, 71.09, 67.88, 63.68,
63.60, 63.29, 62.50, 61.86, 60.58, 59.86, 57.60, 57.32, 57.01, 24.56,
22.56, 16.45, 12.01.
Inhibition Studies: A. r-1,3-fucosyltransferase V. Soluble FucT
V was expressed in the filamentous fungus, Aspergillus niger var.
awamori. The strain was chromosomally integrated with a gene
construct containing the FucT V catalytic domain7i fused to the
glucoamylase promoter and coding region.45 FucT V was purified from
fungal supernatants by 20-60% ammonium sulfate precipitation and
phenyl sepharose column chromatography. Approximately 300 units
of FucT V can be prepared per liter of fermentation solution (1 unit of
enzyme will consume 1 µmol of GDP-Fuc per minute). The enzyme
was stored at -20 °C in 150 mM NaCl, 50 mM MOPS, pH 7.0, 10
mM MnCl2, and 50% glycerol.
The activity of FucT V was detected by the assay described
previously.46 The assay pH was varied from 6 to 8. The pH opti-
mum was found to be 6.2. All assays contained 10 mM MnCl2, 10
mM ATP, 2.1 munit of purified FucT V (except where noted), and
25 mM cacodylate buffer (pH 6.2) in a total assay volume of
0.05 mL. Assays were performed at 25 °C. Reactions were halted
with the addition of 0.5 mL of distilled, deionized water. GDP-
fucose was separated from the product, Lewis x, with a 1.0 mL
Dowex1-X8 pipet column. The reaction mixtures were applied to the
column and washed with 0.3 mL distilled, deionized water three times.
The flow through and the column washes were collected in 10 mL of
ScintiVerse I scintillation cocktail. Control reactions, without enzyme,
were used to establish the background, nonenzymatic cleavage rate. A
typical control reaction of 32 672 cpm guanosine-5′-diphospho-L-[U-
14C]fucose would result in 173 cpm of nonenzymatic column flow
through.
An initial experiment with 24 mM LacNAc and 0.05 mM GDP-Fuc
(0.67 Ci/mol) was used to define the time course of the reaction.
Aliquots (0.05 mL) of the 0.25 mL enzymatic reaction were taken at
60, 90, 155, and 330 min. Time points at 1 h or less were judged to
be in the linear portion of the velocity-time profile. All subsequent
data was obtained with 1 h of reaction time. Kinetic parameters for
R-1,3-fucosyltransferase V were in agreement with the literature values.
Data was subjected to nonlinear least squares fit of the Michaelis-
Menton equation with the Hypero FORTRAN program of Cleland.22
Concentrations of LacNAc were 5-80 mM at 0.05 mM GDP-Fuc. The
Km of LacNAc was determined to be 31.1 ( 6.2 mM while the literature
value is 35 mM.8,9 The 1/Vmax was determined to be 0.0241. The
apparent Km of GDP-Fuc was determined at 24 mM LacNAc and
0.025-0.4 mM GDP-Fuc to be 0.078 ( 0.032 mM. Initial Ki values
were obtained with Dixon analysis at 24 mM LacNAc and 0.05 mM
GDP-Fucose. GDP was evaluated at 0, 0.05, 0.25 mM with 24 mM
LacNAc and 0.025, 0.050, 0.10, 0.20, and 0.40 mM GDP-fucose. The
Ki of Lex was determined by Dixon analysis at 0.05 mM GDP-fucose,
24 mM LacNAc, and variable Lewis x (0, 10, 20, 30, and 40 mM).
23
Lex with a â-O-(CH2)5CO2CH3 at the reducing end (Ki ) 0.125 (
0.0142 mM) was used to determine the mode of inhibition of Lex at 0,
0.4, and 1.0 mM with 0.05 mM GDP-Fuc. This Lex derivative (0, 0.2,
0.4 mmol) was analyzed at 20 mM LacNAc and at variable GDP-Fuc
concentrations (0.025, 0.050, 0.10, and 0.20 mmol) and it did not inhibit
bovine â-1,4-galactosyltransferase. Compound 1 was evaluated at 0,
5, 20, 48, and 90 mM and 2 was evaluated at 0, 1, 2, 3, 6, and 15 mM
by Dixon analysis at 0.05 mM GDP-fucose and 24 mM LacNAc. The
mode of inhibition was established with double reciprocal plots at
inhibitor levels of Ki and greater. Statistical analysis of the fit of the
product inhibition data to the equations for competitive, noncompetitive,
and uncompetitive inhibition was used to confirm the mode of
inhibition. This was accomplished with the Compo (eq 4), Ncomp
Propyl 2-Acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-3-O-(2-
(N-(â-L-homofuconojirimycinyl))ethyl)-r-D-glucopyranoside (2). To
a solution of compound 15 (17 mg, 13.6 µmol) in dry MeOH (2.0 mL)
was added NaOMe (30 µL of a 0.5 M solution in MeOH, 15 µmol).
The resulting mixture was stirred at room temperature for 2.5 h and
then concentrated to give a glassy residue: 1H NMR (CDCl3, 400 MHz)
δ 7.35-7.20 (m, 25 H, ArH), 4.65-4.35 (m, 12 H), 4.15-3.16 (m, 32
H), 1.89 (s, 3 H), 1.43-1.38 (m, 2 H), 1.18 (d, 3 H, J ) 6.8 Hz), 0.75
(t, 3 H, J ) 7.2 Hz); HRMS (LSIMS+) m/z calcd for C61H78N2O15
+
H+ 1079.5480, found 1079.5526.
(45) Dunn-Coleman, N. S.; Bloebaum, P.; Berka, R. M.; Bodie, E.;
Robinson, N.; Armstrong, G.; Ward, M.; Przetak, M.; Carter, G. L.; LaCost,
R.; Wilson, L. J.; Kodama, K. H.; Baliu, E. F.; Bower, B.; Lamsa, M.;
Heinsohn, H. Bio/Technology, 1991, 9, 976.
(46) Sarnesto, A.; Ko¨hlin, T.; Hindsgaul, O.; Vogele, K.; Blaszcyk-
Thurin, M.; Thurin, J. J. Biol. Chem. 1992, 267, 2745.
The residue obtained above was dissolved in a solution of MeOH:
AcOH (2:0.75 mL), and then Pd(OH)2/C (Degussa type, 16 mg) was
added under an argon atmosphere. The mixture was hydrogenated at
1 atm for 18 h. Filtration through a pad of Celite and concentration in
Vacuo gave a residue. Filtration through a Bio-gel P-2 column gave 2

7662 J. Am. Chem. Soc., Vol. 118, No. 33, 1996
Qiao et al.
(eq 5), and Uncomp (eq 6) FORTRAN nonlinear least square programs
adapted for the Apple Macintosh. Compound 2 was evaluated at 0.05
mM GDP-Fuc, variable LacNAc (5, 10, 20, and 40 mM), and fixed 2
(0, 2, 4, 6, 8 mM) and evaluated with the Ncomp (eq 5) FORTRAN
program. Compound 2 was also evaluated at 24 mM LacNAc, variable
GDP-Fuc (0.02, 0.05, 0.1, and 0.2 mM) at fixed 2 (0, 4, 8 mM). The
Ki value was derived from nonlinear, least squares fit to the kinetic
equation for competitive inhibition by the Compo FORTRAN program
(eq 4).
mM). Reactions contained 0.3 munits FucT V and were allowed to
react at 25 °C for 30 min. Mono aza sugars were evaluated in a similar
manner. The inhibition of the following inhibitor(s) was evaluated:
no inhibitors, GDP only, aza sugar only, and the combination of GDP
and an aza sugar. Compound 1, 5-membered fucose-type aza sugar,
trans 5-membered aza sugar, GDP, and GDP-fucose were held con-
stant at 33, 80, 34, 0.05, 0.05 mM respectively. Reactions con-
tained 0.3 munits with the LacNAc concentration varied from 5 to
40 mM.
B. r-L-Fucosidase. Inhibition studies of R-L-fucosidase were
carried out on bovine epididymus R-L-fucosidase monitoring the
hydrolysis of p-nitrophenyl R-L-fucoside in a 50 mM sodium acetate
buffer (pH 6.5) at 37 °C. Liberation of p-nitrophenolate anion was
monitored in a 1.0 mL assay over the course of 2 min at 400 nm on a
Beckman DU-6 spectrophotometer. Determinations were the average
of at least three measures. Enzyme kinetic parameters were calculated
by nonlinear regression analysis with the Hypero program. Inhibition
constants were first estimated by the method of Dixon at a Km level of
substrate.35 The mode of inhibition of 1 was evaluated by double
reciprocal analysis of data derived from monitoring the velocity as a
function of p-nitrophenyl R-L-fucoside (0.075, 0.15, 0.30, 0.60, 1.28
mM) at fixed 1 concentrations (0, 4, 8, 16 nM). A precise inhibition
constant was derived for 1 from a best fit of the initial velocity data to
the kinetic equation for competitive inhibition by a nonlinear, least
squares method, the Compo FORTRAN program (eq 4).
Compo: V ) Vmax*[S]/([S] + Km*(1 + [I]/Ki))
Ncomp: V ) Vmax*[S]/([S](1 + [I]/Ki) + Km*(1 + [I]/Ki))
Uncomp: V ) Vmax*[S]/([S](1 + [I]/Ki) + Km*)
*apparent kinetic constants
(4)
(5)
(6)
Synergistic Inhibition. Synergistic inhibition of was initially probed
by monitoring the percent inhibition of FucT V (0.05 mM GDP-Fuc,
24 mM LacNAc) with the individual inhibitors and combination of
GDP and the aza sugars: 1 (33 mM), 2 (2.5 mM), GDP (0.05 mM),
the combination of 1 and GDP, and the combination of 2 and GDP.
Yonetani-Theorell analysis36 was used next to address synergism of
multiple inhibitors. LacNAc and GDP-Fuc were held constant at 24
and 0.05 mM, respectively. 2 was varied (0, 1, 2, 4 mM) at fixed
concentrations of GDP (0, 0.05, 0.1, 0.25 mM). A steady-state
derivation for the synergistic inhibition of FucT V (Scheme 4) in the
absence of Lewis x was undertaken. The kinetic constants are as
follows: Ki,GDP-fuc ) k-1/k1, km,GDP-fuc ) k3/k1, km,LacNAc ) (k-2 + k3)/
k2 and Vmax ) k3Et. The resulting equation (eq 3) is in a form similar
to the equation found in the literature for inhibition by the second
product released (GDP) in an ordered, sequential BiBi mechanism (eq
1). The data obtained was fitted to eq 3.
Acknowledgment. We thank Professor John Lowe for
providing the FucT V cDNA and Dr. James C. Paulson for
helpful discussions. This work is partially supported by the
NIH (GM 44154) and Cytel Corp. (San Diego, CA).
Supporting Information Available: 1H and 13C NMR
spectra for compounds 1, 2, 7, 8, 9, 12, 13, 14, 15, and inhibition
study of R-fucosidase (17 pages). See any current masthead
page for ordering and internet access instructions.
The role of the acceptor sugar in the synergistic inhibition was
evaluated by monitoring FucT V activity as a function of LacNAc
concentration at fixed GDP-Fuc (0.05 mM), GDP (0.03 mM), 2 (2.8,
14 mM), and the combination of GDP (0.03 mM) and 2 (2.8, 14
JA960274F